NU. International Journal of Science 2016; 13(2) : 49-61 49 Identification of some volatile compounds in rice infestation with brown planthopper (BPH) Panatda Jannoey, 1* Duangdao Channei 2 , Jate Kotcharerk 3 , Weerathep Pongprasert 4 and Tanyarat sutti 1 1,5 Department of Biochemistry, Faculty of Medical Science, Naresuan University, Pitsanulok, 65000, Thailand 2 Department of Chemistry, Faculty of Science, Naresuan University, Pitsanulok, 65000, Thailand 3 Phitsanulok Rice Research Center, Wang Thong, Phitsanulok , 65130, Thailand 4 Department of Agricultural Science, Faculty of Agriculture, Natural Resources and Environment, Naresuan University, Pitsanulok, 65000, Thailand * Corresponding author. E-mail:[email protected]ABSTRACT Rice volatile compounds induced by brown planthopper BPH were analysed using capillary gas chromatography-mass spectrometry (GC-MS).Fifty-three (53) different volatile compounds were found in the rice infested with BPH and the control.Twenty-nine (29) volatile compounds increased emission in the rice infested with BPH, including alkane ( nonane, dodecane, cyclopentane, tetradecane, cyclohexane and nonacosane) , ketone (ethanone), alkene (1-dodecene), alcohol (5-methyl-4,7,10,13-tetraoxatetradeca-2-ol, phytol), aldehyde (E-15-heptadecenal and pyrrole).Gamma and alpha tocopherol, neophytadiene, dibutyl phthalate and stigmasterol were found in the control than those the attacked rice, whereas naptho[2,3-b] furan-4,9-dione was found as a major constituent in resistant cultivar after the BPH infestation.The gene involved in volatile compound production,OsHPL, also increased significantly following BPH infestation. The volatile compounds incorporating the gene may be responsible for the characteristic of rice being resistant to BPH in the resistant cultivar. Keywords: Naptho[2,3-b] furan-4,9-dione, brown planthopper, rice, volatile compounds, metabolites INTRODUCTION The emission of low molecular weight volatile organic compounds (VOCs) is one of the plants’defence responses to pathogens and insects (Zhuang, X. et al.,2012). The plant volatiles function as an indirect defence to pathogens by attracting the natural enemies of their herbivores (Turlings,T.C.J. and Wickens, F.L.,2004;Howe, G.A. and Jander, G., 2008).The parasitoid, Anagrus nilaparvatae, a major natural enemy of the rice BPH, was attracted to rice infested with BPH, whereas there was no attraction to volatiles which were released from undamaged plants (Lou,Y.G., et al., 2005).Among the VOCs, green leaf volatiles (GLV) which are produced from green plants upon herbivory and pathogen infection play an important role in plant defence (Scala, A. et al., 2013).Previous studies revealed that herbivore attack induces a set of defensive responses in rice, activating the signalling pathways (jasmonate and ethylene signalling) by expressing defensive genes (Hao, Z., et al., 2011; Duan, C., et al., 2014;Zhang, F., et al., 2004). For example, rice plants
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NU. International Journal of Science 2016; 13(2) : 49-61 49
Identification of some volatile compounds in rice infestation
with brown planthopper (BPH)
Panatda Jannoey,1*
Duangdao Channei 2, Jate Kotcharerk
3,
Weerathep Pongprasert 4 and Tanyarat sutti
1
1,5 Department of Biochemistry, Faculty of Medical Science,
Naresuan University, Pitsanulok, 65000, Thailand 2 Department of Chemistry, Faculty of Science, Naresuan University,
Pitsanulok, 65000, Thailand 3 Phitsanulok Rice Research Center, Wang Thong, Phitsanulok , 65130, Thailand
4Department of Agricultural Science, Faculty of Agriculture, Natural Resources
and Environment, Naresuan University, Pitsanulok, 65000, Thailand
50 NU. International Journal of Science 2016; 13(2) : 49-61
damaged by the feeding of the Spodoptera frugiperda (FAW) were shown to emit a blend of volatiles that are highly attractive to parasitic wasps (Yuan, J.S. et al.,
2008).
Recently, Wang et al. (2015) reported that herbivore-induced plant VOCs play
an important role in regulating interactions between plants, herbivores and their
natural enemies. Moreover, the production of rice volatiles induced by BPH showed
an equal attraction of the parasitoid (Yujie, L. et al., 2006).
The BPH, Nilaparvata lugens Stål (Hemiptera: Delphacidae), is a destructive
insect pest of rice, causing reductions in rice yield and large economic losses
(Jannoey, P. et al., 2015; Jannoey, P and Channei, D., 2015). BPH outbreaks occur in
tropical regions, especially in the central and lower northern regions of Thailand.
The outbreak areas were observed in seven provinces, Sukhothai, Phitsanulok,
Chai Nat, Ang Thong, Suphan Buri, Nonthaburi and Nakhon Nayok, during the
raining season. The BPH suck sap in the seedling and tender spike stage, resulting in
yellowness, wilting and even death, these symptoms are called hopperburn.
Outbreaks of BPH in Thailand had a significant impact during the last four years
(2012-2015), causing the loss of 2.68, 1.68 and 1.18 million hectares of the rice area,
respectively. The economic loss in the last four years was more than 26,000 million
baht.
VOCs have been reported to be a messenger protecting plants against herbivores
and pathogens. A recent study showed that (E)--caryophyllene, one of the VOCs,
has been reported to attract BPH and its egg parasitoid, A. nilaparvatae (Wang, Q.
et al., 2015a).Cheng, A.X. et al. (2007) found that the transgenic plants of both rice
and Arabidopsis with higher levels of rice (E)--caryophyllene synthase (OsTPS3)
attracted more wasps of A. nilaparvatae Pang et Wang, a main egg parasitoid of rice
BPH.
The main volatiles in rice constituent are indole (Zhuang, X. et al., 2012), terpene
including cinnamyl alcohol, myristicin, sesquiterpene alcohol (caryolan-1-ol),
(E)--caryophyllene, sesquiterpenes (Forlania, G. et al., 2011; Wang,G. et al., 2015;
Cheng, A.X. et al., 2007), alkanes, alkenes and long-chain alcohols (Forlania, G. et al., 2011) has been reported. Zhuang, X. et al. (2012) found that the indole
synthesised by indole-3-glycerol phosphate lyase was emitted from rice-herbivory
induction. Similarly, rice plants damaged by FAW (Spodoptera frugiperda) larvae
emitted about 30 volatiles, including methyl salicylate and methyl benzoate. FAW-induced volatiles are highly attractive to female parasitic wasps (Cotesia
marginiventris),carnivorous enemies of FAW (Zhao, N. et al., 2010). Furthermore, the VOC included alkanes, alkenes and long-chain alcohols as well
as cinnamyl alcohol, myristicin, sesquiterpene alcohol (caryolan-1-ol), 1-octanol and
1-decanol showed fungistatic activity against to M. oryzae (Forlania, G. et al., 2011).
Interestingly, the hydroperoxide lyase (OsHPL) gene is one of the genes encoded
for hydroperoxide lyase (HPL) enzyme production.The HPL enzyme is the key
enzyme for green volatiles biosynthesis, its activity represents a key function of
volatile compounds in plants as an indirect defence.The expression of the OsHPL
gene in Arabidopsis caused increased volatile compounds production, resulting in
higher attraction of parasitic wraps to control their herbivores. The loss of mutant
Os-HPL3 function in rice showed lower VOCs emission (Scala, A. et al., 2013).
NU. International Journal of Science 2016; 13(2) : 49-61 51
Although the release of herbivore-induced plant volatiles has been reported
above, there is a little evidence that the volatile induced by BPH biotype in Thailand.
Indeed, the volatile and BPH interaction are very few data in resistant cultivar.
The aim of this study was to analyse and identify volatile-induced BPH and HPL
gene expression in resistant cultivar. Gas chromatography (GC) coupling
with Mass spectrometry (MS) technique and Real-time PCR were employed to
analyse volatile compounds and HPL gene expression, respectively. The volatile-
induced BPH may function as an important signal for understanding rice and BPH
interaction.
MATERIAL AND METHODS
Rice plant growth and insect treatments
Rice seeds (O. sativa sp. Indica) were germinated and grown in a greenhouse for
two weeks. BPH were cultivated in a cage with rice seedlings as a food source until
they hatched. The BPH nymphs were transferred to rice plants in the caged condition.
Rice plants were infested with BPH in a ratio of 12:1 insects per rice.
Rice phenotypes were monitored and collected for volatile compound analysis
compared to the rice without BPH infestation (control).
Volatile compound analysis
GC conditions
Analysis of the volatiles of rice seedlings was performed by using 600 mg of
tissues ground in liquid nitrogen. Rice samples were extracted with dichloromethane,
centrifuge at 6,000 rpm for 30 minutes. The crude extract was collected in a 1.5 ml
vial and sealed with a magnetic cap. The samples were placed in an autosampler tray
and were maintained at room temperature until analysed. The initial temperature was
held at 250oC using HP-5MS capillary column (0.25mm×30m×0.25µm; Agilent
19091S-433).The GC oven temperature was held for 1 minute at 250oC, then
increased to 260oC for 30 minutes at a rate of 5
oC/minute in splitless mode.
MS conditions
The interface temperature was 280°C, the ion source using the Electron Impact
(EI) mode at 230°C, the electron energy was 70 eV and the mass scan range (M/Z)
was 35-350 amu.
OsHPL gene expression analysis
RNA extraction and cDNA synthesis
The total RNA was extracted with E.Z.N.A.® Total RNA Kit (Omega) according
to the manufacturer’s instructions and then quantified using a NaNoDrop ND-100
spectrophotometer.The total RNA was converted to cDNA using Tetro cDNA
Synthesis Kit (Bioline).The cDNA synthesis reactions were incubated at 37oC for
15 minutes and 85oC for 5 seconds. The cDNA was obtained and then amplified in
a further step.
Quantitative real-time PCR
Quantitative real-time PCR was performed using SensiFAST™ SYBR® Kits
(Bioline). The PCR reaction was carried out in triplicate in 48-well plates. The thermal
profile of the real-time system was set as one step at 95oC for 30 seconds, followed by
40 cycles at 95oC for five seconds (denaturation) and 60
oC for 30 seconds (annealing
52 NU. International Journal of Science 2016; 13(2) : 49-61
and extension), followed by added dissociation pattern. The actin gene was used as
the internal standard.
Data analysis
The quantification of the relative expressions level was determined from the
average threshold cycles (CT).The CT value was calculated by subtracting the
average CT value of the interested gene from the CT value of the actin gene.
2- CT
was calculated to estimate the relative expression level.
RESULTS AND DISCUSSION
Rice phenotype during BPH infestation
Rice seedlings were infested with BPH nymphs, and hopperburn symptoms were
observed on different infestation days (Table1).Following infestation, continuous
feeding by BPH nymphs caused wilting of the seedlings, leading to hopperburn
(browning of stem and leaves) (Figure 1).During early infestation (0-7 days after
infestation, DAI), damage symptoms were not detected on infested plants except
the TN1 cultivar. This is probably due to the fact that the BPH did not cause enough
damage and plants were able to overcome the low level of insect stress (Sangh, J.S. et
al., 2013) and some cultivars contained BPH-resistant genes.The difference in the
phenotype among the cultivars was more obvious at 8-15 DAI, respectively.
However, the results in Table 1 categorised the rice’s resistance to BPH ability into
three groups.The highest resistance to the BPH cultivar was the 67-111, PTB33,
Rathu Heenati, PSL2 and RD49 cultivars, whereas RD41 and RD47 were categorised
into moderately BPH-resistance ability. Finally, the susceptible cultivar was found in
TN1, SP90 and PT1. Therefore, PTB33 was chosen to extract to identify OsHPL gene
and the total volatile compounds in this study. The identified metabolites may be
useful for understanding the mechanism involved in the rice’s resistance to BPH
in the resistant cultivar.
Comparison of volatile compounds emitted from rice-treated BPH
The volatile compounds analysis was collected from dichloromethane fraction.
Fifty-three different components were found and classified on the basis of their
cyclohexane, undecane, hexadecane, 2,5,8,11,14,17-hexaoxaoctadecane and
nonacosane) were found to be the most abundant components, followed by alkene (1-dodecene), ketone (ethanone), aldehyde (E-15-Heptadecenal) and ester (1-methylethyl tetradecanoate). Moreover, neophytadiene, dibutyl phthalate, 2H-
anti-diabetic, antioxidant and radical scavenging activity (Venkata, R.B. et al., 2012). However, these compounds reduced emission in rice after BPH infestation, Similarly, Lou et al. (2005) found that the BPH infestation induced increasing
of the tetradecane, pentadecane and linalool, subsequently attractive to the BPH’s
natural enemy. Moreover, previous reports found that most compounds of heptanone, 2-heptanol, limonene, linalool and (E)-4,8-dimethyl-1,3,7-nonatriene were released in
NU. International Journal of Science 2016; 13(2) : 49-61 53
significantly higher amounts from BPH-JA-treated rice plants compared to the
control. The volatiles emitted from JA-treated rice plants attracted the BPH to the
parasitoid and enhanced the parasitism of N. lugens eggs in the greenhouse and the
field (Thaler, J.S. 1999; Kessler and Baldwin, 2001). Furthermore, it was previously
shown that linalool is attractive to A. Nilaparvatae. A difference in the attractiveness
of linalool to BPH was found between JA-induced plants compared to the buffer
solution. However, Wang, Q. et al. (2015) suggested that the rice white-backed
planthopper (Sogatella furcifera) preferred feeding and ovipositing on wild-type
plants to feeding on mutants with low levels of (E)--caryophyllene,
whereas the other studies indicate that (E)--caryophyllene may function as an
important signal by which herbivores on rice locate their host.
In contrast with the previous reports, GLV have not been found in this study.
GLV constituents are composed of E-2-hexenal, E-2-hexenol, E-2-hexenal-acetate,
Z-3-hexenal, Z-3-hexenol, Z-3-hexyl acetate, n-hexanal, n-hexanol and n-hexenyl
acetate. These compounds are semi-chemicals used by insects to find their food
source, while plants use GLV as an indirect defence and a direct effect on pests
(Scala, A. et al., 2013). Many studies have shown that the predator and parasitoids are
indeed attracted to GLV. For example, the aliphatic aldehyde (hexanal and E-2-
hexenal) attract female BPH more than the other volatile compounds including leaf
alcohol (Youan, N.Y. 2002). The previous report by Qiang et al. (2003) showed that
the GLV compounds (E-2-hexenal, E-2-hexen-1-ol) had a significantly repellent
effect on the adult BPH at higher concentration, resulting in increased susceptibility
of rice to white-black BPH. Other reports found, however, that the E-2-hexen-1-ol, 2-
heptanone, limonene, ocimene, linalool, -caryophyllene and (E)-nerolidol
had no significant repellent effect on BPH (Qiang et al., 2003).
Naphtho[2,3-b]furan-4,9-dione was only found in resistant cultivar due to BPH
attack in this study (Figure 2). It is an important bioactive compound extracted from
the trunk and inner bark of Tabebuia billbergii (Gómez-Estrada, H. et al., 2012;
Ogawa,M. et al., 2009). Ogawa, M. et al. (2009) reported that several naphtho[2,3-
b]furan-4,9-diones isolated from plants and synthesized having biological activities.
For example, 2-acetylnaphtho[2,3-b]furan-4,9-dione isolated from Tabebuia
cassinoides (Lam.) DC (Bignoniaceae) exhibited cytotoxic activity of tumour-specific
cytotoxicity. The authors suggested that the hydroxyl radical was the active
intermediate of these agents involved in the cytotoxic mechanism of naphtho[2,3-
b]furan-4,9-diones. Moreover, the naphtho-furan-diones showed important inhibitory
activity when assayed in vitro against Plasmodium berghei, especially in comparison
to the activity exhibited by chloroquine, which was used as the control substance
(Gómez-Estrada, H. et al., 2012). These compounds has been reported as a fungicides
(botanical fungicides), “santonin” is approved as a common name in China.
Naphtho[2,3-b]furan-4,9-diones is also related to a mixture of novel pyrazole
compounds formula for combatting invertebrate pests such as insects, arachnids
or nematodes on plants, preventing such plants being infested with pests (WO
2013189801 A1). It is possible that naphtho[2,3-b]furan-4,9-dione may play
a role in protecting rice plants from BPH attacks. Therefore, this finding indicates
that the volatile compounds emitted from rice treated BPH may be responsible for
BPH defence mechanism in resistant cultivar. However, the volatile compounds
against BPH mechanism (indirect or direct) had not been proved in this study.
60 NU. International Journal of Science 2016; 13(2) : 49-61
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